This Academic Research Enhancement Award (AREA) R15 application is focused on the molecular and biochemical mechanisms of viral RNA synthesis and mRNA posttranscriptional modifications in nonsegmented negative-strand (NNS) RNA viruses (taxonomic order Mononegavirales). This order includes a wide variety of human, animal and plant viruses, in which the RNA polymerase complex possesses unique features. The proposed research aims to examine the role of the virus-encoded large (L) polymerase protein in viral RNA synthesis and mRNA posttranscriptional modifications. This protein has six sequence regions ("domains") with a high degree of homology among all Mononegavirales, which have been postulated to constitute specific enzymatic activities of the viral RNA polymerase involved in transcription, mRNA capping, methylation of 54 cap structures, polyadenylation, and replication of viral genomic RNA. Despite its importance, the L protein is not well characterized, and studies directed at identifying specific functions of these regions have been limited. The specific aims are: 1) Performing a comparative analysis of the L protein cap methylation function in rhabdo- and paramyxoviruses; 2) Defining the relationship between the two L protein regions involved in cap methylation; 3) Revealing the mechanism of mRNA cap methylation- mediated host restriction in Mononegavirales; and 4) Characterizing the L protein sequences important in mRNA polyadenylation. The studies will employ two prototypic Mononegavirales: vesicular stomatitis virus (VSV, a rhabdovirus) and Sendai virus (SeV, a paramyxovirus), which serve as attractive models due to their robust replication in a wide range of cell lines, well-established in vitro systems for the study of RNA synthesis, and available reverse genetics systems. This two-virus approach will allow for the identification of universal as well as virus-specific L protein activities within this group of viruses, because VSV and SeV belong to different families. These studies should provide new clues for understanding the molecular mechanisms of viral RNA synthesis and for developing new ways to combat viral infections. PUBLIC HEALTH RELEVANCE: This application is focused on the molecular and biochemical mechanisms of mRNA posttranscriptional modifications in nonsegmented negative-strand RNA viruses (taxonomic order Mononegavirales). This order contains many lethal human pathogens, including the Ebola and Marburg viruses which are classified as category A bioweapon agents by the Centers for Disease Control and Prevention, and highly prevalent human pathogens, such as the respiratory syncytial and parainfluenza viruses. The L protein of Mononegavirales has been postulated to catalyze all enzymatic activities of the viral RNA polymerase involved in transcription, mRNA capping, methylation of 54 cap structures, polyadenylation, and replication of viral genomic RNA. Despite its importance, the L protein is not well characterized, and studies directed at identifying specific functions of this protein have been limited. The proposed studies are important to define the basic mechanisms of reproduction of these viruses, with the ultimate hope that this knowledge will contribute not only to the basic understanding of RNA macromolecular biosynthetic pathways, but will also lead to new or better therapeutic agents. [unreadable] [unreadable] [unreadable]